JP2001031700A - New antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To find a causal substance for an unknown apoptosis avoidance mechanism, to provide a means for controlling the causal substance and to provide a means for the prophylaxis and therapy of cancer and also a cancer-diagnosing means using the causal substance as a marker. SOLUTION: An antibody carrying apoptosis inducing power of a cancer cell recognizes a glycoprotein antigen expressed strongly on cell membrane of human colon cancer cell line colo 205 cell irrespective of presence or absence of interferon-γ stimulation and existing in an epithelial cell of human normal tissue. A compound is identified utilizing a polypeptide immunologically recognized by the antibody, a polynucleotide encoding the polypeptide or reactivity with the polypeptide and/or polynucleotide. A means for controlling an apoptosis system in a living body by utilizing them is provided, a new means for the prophylaxis and therapy of cancer is obtained, and a new method for diagnosing cancer is obtained using them as markers.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to antibodies, polypeptides, polynucleotides and compounds derived therefrom, which are involved in apoptosis of cancer, especially colorectal cancer, and pharmaceutical compositions containing these, which are useful for the prevention and treatment of cancer. And cancer diagnostic means.

[0002]

BACKGROUND ART As a mechanism of cell death, a mechanism of apoptosis attracts attention, and application of this apoptotic mechanism to cancer treatment is expected. It is reported that what triggers this apoptosis is a substance called Fas antigen, which is a receptor molecule present on the cell membrane.
However, it has been reported that human colon cancer cells show Fas resistance and do not induce apoptosis despite expressing the Fas antigen on the cell membrane (C
ancer Research, 1998, 58:52.
6-534). Therefore, it is presumed that some unknown apoptosis avoidance mechanism still exists in cancer cells.

[0003]

SUMMARY OF THE INVENTION The problem to be solved by the present invention is to search for an unknown apoptosis avoidance mechanism, find a causative agent thereof, and provide a means for controlling the causative agent to prevent cancer. And a means for treatment, and a means for diagnosing cancer using a causative substance as a marker.

[0004]

Means for Solving the Problems The present inventor has previously proposed that interferon-γ (hereinafter abbreviated as IFN-γ) is used to transform human floating colon cancer cell line colo205 cells (hereinafter simply referred to as colo205 cells). It was found that the cell aggregation phenomenon was induced. When the inhibitory effect of this cell aggregation phenomenon was examined using monoclonal antibodies against various cell adhesion molecules, it was partially inhibited only by the anti-CEA antibody, but was not completely inhibited (Cancer Letters). , 1993, 71:
109-117). The present inventors have attempted to create a novel monoclonal antibody using this bioassay system. As a result, a monoclonal antibody having completely novel characteristics was successfully prepared using cells of colo205. The antigen recognized by this antibody was presumed to have a regulatory function on cell apoptosis, and the antibody itself acted on this antigen, and as a result, induced apoptosis of cancer cells. In addition, F, which is conventionally known as an apoptosis-related factor,
It has been reported that the expression of as antigen is enhanced by treating cells with IFN-γ (J. Exp.
Med. 1989, 169: 1747-1756).
However, the antigen recognized by the antibody of the present invention
There was no change in expression after N-γ treatment. Furthermore, it was confirmed that the degree of expression of the antigen recognized by the antibody of the present invention, the Fas antigen known as an apoptosis-related factor, and the tumor necrosis factor receptor (hereinafter abbreviated as TNF-R) differs depending on the cell line. Was. That is, the antigen recognized by the antibody of the present invention is a Fas known as an apoptosis-related factor.
It was different from antigen or TNF-R.

The antigen recognized by the antibody of the present invention is presumed to be deeply involved in cancer cell apoptosis.
The presence of the antigen has value as a novel marker for cancer diagnostic means.

The present invention provides a glycoprotein antigen having the following properties a) and b); a) highly expressed on the cell membrane of human colon cancer cell line colo205 cells regardless of the presence or absence of interferon-γ stimulation; ) An antibody that recognizes mainly present in epithelial cells in human normal tissues, 1) having a concentration-dependent growth inhibitory effect on colo205 cells, 2) inducing DNA fragmentation of colo205 cells, 3) ) Nuclear fragmentation and enrichment of colo205 cells. 4) Colon cancer cell lines HT-29, Caco-2, A
CM, KATO-III, a gastric cancer cell line, MCF-7, a breast cancer cell line, R, a B cell lymphoma cell
5) The present invention provides a novel monoclonal antibody that retains cancer cell-inducing ability and eliminates Fas antigen from colo205 cells after immunological reaction with the antibody.

Further, the present invention provides a polypeptide immunologically recognized by the above-mentioned antibody and a polynucleotide encoding the polypeptide.

[0008] The present invention has the following functions, which have reactivity with this polypeptide and / or the polynucleotide of claim 3 and are at least equivalent in biological evaluation to the antibody of claim 1, 1) against colo205 cells. Has a concentration-dependent growth inhibitory effect, 2) induces DNA fragmentation of colo205 cells, 3) causes nuclear fragmentation and enrichment of colo205 cells,
And a method for identifying a compound carrying the same.

The present invention provides a pharmaceutical composition useful for the prevention and treatment of cancer, which comprises at least one of the antibody, polypeptide or polynucleotide of the present invention or the above-identified compound. I do.

[0010] The present invention provides a means for diagnosing cancer, which comprises using the antibody of the present invention and / or a polypeptide immunologically recognized by the antibody and / or a polynucleotide encoding the polypeptide as a marker. I will provide a.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION (New Antibody) The antibody of the present invention utilizes a colo205 cancer cell line as an immunogen, as shown in the Examples below. However, when the tissue distribution of the corresponding antigen was examined using the obtained antibodies,
The immunogen is not limited to this because it is widely distributed in human normal tissues. The characteristics of the antibody of the present invention are summarized below.

1) The immunoglobulin class confirmed as a monoclonal antibody is mouse IgM. However, monomeric IgG can be used as long as the same immunoreactivity as the antibody of the present invention is maintained. 2) The corresponding antigen is strongly expressed on the cell membrane of colo205 cells, with or without IFN-γ stimulation. 3) No corresponding antigen is found in normal human tissues other than epithelial cells in immunohistochemical studies. 4) It has a growth inhibitory effect on colo205 cells in a concentration-dependent manner. 5) Induce DNA fragmentation of colo205 cells. 6) Nuclear fragmentation and enrichment of colo205 cells. 7) Colon cancer cell lines HT-29, Caco-2, A
CM, KATO-III, a gastric cancer cell line, MCF-7, a breast cancer cell line, R, a B cell lymphoma cell
There is immunological reactivity with aji cells. 8) colo205 after immunological reaction with antibodies of the invention
Cells lose staining with the Fas antigen. That is, the Fas antigen is lost from the cell membrane.

The antibodies of the present invention have been established as monoclonal antibodies. As described above, the technique is to use cancer cells as an immunogen and administer them to animals such as mice, rats, rabbits, goats, horses, and the like, and if necessary, in the presence of an adjuvant, to produce a humoral response and / or a cellular response. Immunity induction is performed. An antibody-producing cell represented by a spleen cell is recovered from the animal to which the inducing means has been applied, and a transforming means is introduced by fusing with a perpetually proliferating cell such as a myeloma cell known per se or transforming into a permanent cell by virus infection. I do. Selection of the antibody-producing cells according to the present invention is based on colo2 reported previously by the present inventors.
Of the bioassay using the cell line 05 (Canc)
er Letters, 1993, 71: 109-11.
Use 7). That is, cells producing an antibody having the cell death-inducing activity of the colo205 cell line were screened, and cloning was repeated by limiting cloning by a known limiting dilution method.

The antibody can be recovered by a method known per se, for example, alcohol, ammonium sulfate, PEG, or the like from a culture solution of the cloned antibody-producing cells or ascites of the mouse into which the antibody-producing cells have been transplanted.
And the like can be used in combination as desired. In addition, it is also possible to suitably isolate using affinity chromatography utilizing specific binding to the antibody of the present invention. Means for obtaining a substance that specifically binds to the antibody of the present invention include, for example, immobilizing the antibody of the present invention,
It is possible to easily find a polypeptide that retains immunological reactivity with the antibody of the present invention by randomly adding a synthetic polypeptide thereto.

By constructing the affinity carrier, polyclonal antibodies having the same specificity as the antibody function of the present invention can be easily recovered from animal serum and the like.

The properties of the antibodies of the present invention have been described in detail in the examples described below.

The polypeptide immunologically recognized by the antibody of the present invention has at least eight amino acid sequences and has at least immunological reactivity with the monoclonal antibody of the present invention. If a cell-free protein synthesis system is used as a means for obtaining this polypeptide, it can be easily recovered. For example, using a ribosome system derived from wheat germ, various mRNAs are prepared using a gene library derived from colo205 as a raw material, and the method of Ericsson et al. (Methods in Enzymology, 19).
83, 96: 38-50), and the produced polypeptide is selectively captured by the antibody-immobilized carrier of the present invention, and then a peptide capable of binding to the antibody, that is, a peptide having antigenicity This can be achieved by identifying and analyzing a polypeptide and a polynucleotide encoding the polypeptide. The polynucleotide of the present invention naturally includes the complementary strand thereof.

Thus, if the antibody of the present invention, the polypeptide having reactivity with the antibody and the polynucleotide encoding the polypeptide can be identified, the biological evaluation can be at least compared with the action of the antibody of the present invention. The following functions are equivalent: 1) having a concentration-dependent growth inhibitory effect on colo205 cells; 2) col
o205 cells to induce DNA fragmentation and 3) colo2
It is possible to provide a method for identifying a compound having a function of causing nuclear fragmentation and concentration of 05 cells. The identification method can be achieved, for example, by screening candidate compounds selected using the reactivity with the polypeptide as a marker, based on the ability to act on colo205 cells by a bioassay established separately by the present inventors. In addition, selection of antagonists or inducers, etc. by drug design techniques based on the sequence or tertiary structure of the polypeptide, selection of expression regulators at the gene level using a protein synthesis system, selection of antibody recognizing substances using antibodies, etc. Can be used in a drug screening system known per se.

The antibody of the present invention thus obtained,
The polypeptides, polynucleotides and identified compounds allow for the modulation of normal cancerous cells or the control of cancerous cells, and compositions containing at least one of these can prevent cancer by a novel mechanism. And a pharmaceutical composition useful for therapy and gene therapy.

Further, the antibodies, polypeptides and polynucleotides of the present invention provide a completely novel diagnostic tool as a marker for canceration of normal cells. Antigens having immunological reactivity to the antibodies of the present invention are widely distributed also in normal cells and found to be unique to epithelial cells, and their expression on cell membranes due to canceration Tended to decrease. For example, expression is high in adenomas and well-differentiated cancers, but low in poorly differentiated cancers. In addition, since this antigen was presumed to be involved in the control of cell apoptosis, it is considered that this antigen may be related to the old and new rotation of the cell in normal cells. Therefore, assaying the variation in the quantity and quality of this antigen by tissue analysis provides a powerful diagnostic means for canceration of cells. Polynucleotides also provide a powerful diagnostic tool for canceration of cells at the genetic level.

【Example】

(Immunization and Cell Fusion) BALB / c mice (6 weeks old, female) were immunized intraperitoneally with 2 × 10 ⁷ cells / 0.1 cc PBS using colo205 cells as an antigen. Three
After immunization three times at weekly intervals, the spleen was removed on the third day, and the spleen cells were fused with mouse myeloma cells Sp2 / 0 using polyethylene glycol 2000 by a method known per se to obtain fused cells (Nature). , 1975, 256: 495-.
497). The obtained fused cells were cultured, and the culture supernatant was screened using a measurement system (bioassay) by observation under a phase-contrast microscope using colo205 cell death as an index.

(Screening using colo205 cell death as an index (bioassay method)) 1 × 10 ⁴ colo205 cells were measured in a 96-well plate for measurement (total 1920).
(Well), the culture supernatant of the fused cells was added, and the cells were cultured for 48 hours, and observed under a phase-contrast microscope to determine the viability of colo205 cells. As a result, one culture well containing the fused cells having the activity of inducing colo205 cell death was obtained. The fused cells were cloned by repeating the limiting dilution method (0.3 cells / well) three times.

(Purification of Antibody) The obtained cloned fusion cells were cultured, and ImmunoAssist was prepared from the culture supernatant.
The antibody was purified using MG-PP (Kanto Chemical Co., Ltd.). The obtained antibody is hereinafter referred to as LHK antibody. The LH
When the immunoglobulin class of K antibody was examined, Ig
M.

(Expression of Antigen Recognized by LHK Antibody on the Surface of Various Cancer Cells) The expression of the antigen recognized by the LHK antibody on the surface of human cancer cells was determined by flow cytometry (FAC).
Scan ^R , Becton-Dickinson) was used for analysis by a standard method. In addition, Fa, which is a cell surface antigen conventionally known to induce apoptosis,
Comparison with s antigen and tumor necrosis factor receptor (TNR-R) expression. 1 μg of LHK antibody, 1 in 10 ⁶ cells
μg of anti-Fas antibody (mouse IgM, Coulter
1 μg anti-TNF-R antibody (hamster IgG,
Coulter) and incubated at 4 ° C. for 30 minutes. The cells were then washed, and LHK antibody, anti-Fas
Cells incubated with the antibody were treated with FITC (fluo
using goat anti-mouse IgM anti-conjugated with anti-TNF (rescein isothiocyanate)
Cells incubated with -R antibody were stained with a goat anti-hamster IgG antibody conjugated to FITC and measured using flow cytometry. As shown in Table 1, colon cancer cell lines HT-29, Caco-2, W
iDr was found in KATO-III, a gastric cancer cell line. Although not shown, the breast cancer cell line MCF
-7, also found in Raji cells, which are B cell lymphoma cells. Furthermore, the distribution of Fas antigen and TNF-R in various cell lines was different from the antigen recognized by the LHK antibody (hereinafter referred to as LHK). In the table, ++ indicates that 10 to 100% of positive cells, + indicates 1 to 10%, and-indicates 0%.

[Table 1] Furthermore, colo205 cells were cultured in the presence of 100 U / ml of human recombinant IFN-γ for 48 hours, and the expression of LHK, Fas antigen, and TNF-R on the cell surface was compared. As shown in FIG. 1, the expression of Fas antigen was enhanced by IFN-γ treatment, but LHK did not change at all. In addition, after colo205 cells were treated with the LHK antibody at 1 μg / ml for 30 minutes at 4 ° C., the expression of Fas antigen on the cell surface was measured by flow cytometry in the same manner as described above. Was done.
Raji cells, which retain LHK on cells but cannot induce apoptosis with LHK antibodies, did not lose the Fas antigen even after similar treatment. From the above results, it was found that LHK is different from the Fas antigen and TNF-R which are conventionally known as apoptosis-related factors.

(Expression of Antigen Recognized by Antibody of the Present Invention in Normal Human Tissues) To confirm the expression of the antigen recognized by the antibody of the present invention in normal human tissues, immunohistochemistry of human frozen tissue sections was performed. The objective staining was performed by a standard method. The antibodies of the present invention were applied to frozen tissue sections and stained by the indirect immunoperoxidase method. As shown in Table 2, in human normal tissues, the antigen recognized by the antibody of the present invention was present in epithelial cells, and was not found in any other cells.

[Table 2]

(Suppression of proliferation of colo205 cells by LHK antibody) The activity of suppressing proliferation of colo205 cells by the LHK antibody was measured. Ju, which retains LHK on the cell surface but cannot induce apoptosis with LHK antibodies
rkat cells compared to anti-Fas antibody or T
It was compared with the growth inhibitory activity by NF. 1 × 10 ⁴ cells
The cells were cultured for 48 hours together with the LHK antibody, anti-Fas antibody and TNF, and further cultured for 12 hours after adding [ ³ H] thymidine, and the radioactivity incorporated into the cells was measured. As shown in FIG. 2, the proliferation of the colo205 cells was significantly suppressed by the LHK antibody, but not by the anti-Fas antibody and TNF. On the other hand, Jur which has LHK on the cell surface but cannot induce apoptosis with LHK antibody
The proliferation of kat cells was suppressed by anti-Fas antibody and TNF, but LHK antibody had no effect. That is, LHK is a known apoptosis-related factor, F
As and TNF-R were found to be different cell death inducing factors.

(Induction of DNA Apoptosis by LHK Antibody) c treated with 1 μg / ml of LHK antibody for 12 hours
DNA was extracted from olo205 cells by a conventional method (J. Immunol., 1987, 139: 3199).
-3206), DNA fragmentation was measured. As shown in FIG. 3, cells treated with the LHK antibody clearly showed DNA
Fragmentation was confirmed. In addition, colo205 cells similarly treated with the LHK antibody were stained with Hoechst 33258 and measured under a fluorescence microscope. As a result, fragmentation and enrichment of nuclei were confirmed. That is, it was found that the LHK antibody induces cell apoptosis.

(Confirmation of LHK) c recognized by LHK antibody
For confirmation of antigen LHK on the cell surface of
olo205 cells (5 × 10 ⁶ cells) were labeled with biotin, and the cells were lysed with a lysis buffer.
Centrifuge at 00 rpm for 20 minutes. The supernatant was subjected to LHK antibody-
Reacted with anti-mouse IgM antibody-agarose beads.
After washing five times with a buffer, the sample was dissolved in a sample buffer, reacted at 95 ° C. for 1 minute, and electrophoresed. After electrophoresis, P
Transfer to a VDF membrane and use a blocking buffer for 3
Allowed to react for hours. After washing, the PVDF membrane was reacted with an ABC kit. After the reaction, the plate is washed and an ECL kit (ECL
Detection System: Amersha
mnbucks, UK) and developed. As a result, as shown in FIG.
A kDa glycoprotein was identified.

As described above, the antibodies, polypeptides, polynucleotides, and identification compounds of the present invention are related to the regulation of novel apoptotic mechanisms, and a means for controlling the apoptotic system in a living body by using them. To provide a novel means for preventing and treating cancer, and a novel means for diagnosing cancer using these as markers.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the results of measurement by flow cytometry of changes in the expression of LHK, Fas antigen, and tumor necrosis factor receptor (TNF-R) on the cell surface due to culture treatment with addition of interferon-γ (IFN-γ). FIG. In the figure, I
FN-γ (-) is cultured without interferon,
IFN-γ (+) means interferon-added culture.

FIG. 2. LHK antibody, anti-Fas antibody, TNF, Co
It is a figure which shows the growth suppression activity in lo205 cell and Jurkat cell.

FIG. 3. Colo2 caused by LHK antibody
5 is a photograph showing DNA fragmentation of 05 cells.

FIG. 4 is a photograph in which LHK present on the cell surface of colo205 cells was confirmed by immunoprecipitation using an LHK antibody.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI theme coat ゛ (Reference) C12N 15/02 G01N 33/574 Z G01N 33/574 C12P 21/08 // C12P 21/08 C12N 15/00 C (72) Inventor Mamoru Watanabe 35 Shinanomachi, Shinjuku-ku, Tokyo Keio University Keio Cancer Center F-term (reference) 4B024 AA01 AA11 BA45 DA02 GA03 GA18 GA23 HA15 4B064 AG27 CA10 CA20 CC24 DA01 DA14 4C084 AA02 AA07 BA44 DA39 ZB212 ZB262 4C085 AA13 AA14 BB01 BB11 DD61 4H045 AA10 AA11 AA30 BA53 CA41 DA76 DA86 EA28 EA51 FA72 GA15 GA26 HA06

Claims (7)

[Claims] 1. a glycoprotein antigen having the following properties a) and b): a) strongly expressed on the cell membrane of a human colon cancer cell line colo205 cell with or without stimulation with interferon-γ, b) human An antibody that recognizes mainly present in epithelial cells in normal tissues, 1) has a concentration-dependent growth inhibitory effect on colo205 cells, 2) induces DNA fragmentation of colo205 cells, 3) colo205 Nuclear fragmentation and concentration of cells are performed. 4) Colon cancer cell lines HT-29, Caco-2, A
CM, KATO-III, a gastric cancer cell line, MCF-7, a breast cancer cell line, R, a B cell lymphoma cell
5) an antibody that retains the ability to induce apoptosis of cancer cells, which is capable of eliminating Fas antigen from colo205 cells after immunological reaction with the antibody. 2. A polypeptide immunologically recognized by the antibody of claim 1. 3. A polynucleotide encoding the polypeptide of claim 2. 4. It has reactivity with the polypeptide according to claim 2 and / or the polynucleotide according to claim 3, and has at least the following functions equivalent in biological evaluation to the antibody according to claim 1. On the other hand, has a concentration-dependent growth inhibitory effect, 2) induces DNA fragmentation of colo205 cells, and 3) causes nuclear fragmentation and enrichment of colo205 cells,
A method for identifying a compound carrying the compound. 5. A compound identified by the method of claim 4. 6. A pharmaceutical composition useful for the prevention and treatment of cancer, which comprises at least one of the substances according to claims 1-3. 7. The antibody of claim 1 and / or 2.
A cancer diagnostic means, which comprises using the polypeptide of (1) and / or the polynucleotide of (3).